JPWO2010024289A1 - IL-17 producing helper T cell marker and method for detecting IL-17 producing helper T cell - Google Patents

Abstract

The present invention comprises detecting at least one polynucleotide marker or protein marker that enables specific detection of IL-17 producing helper T cells (Th17 cells) and the presence of at least one said marker. The present invention relates to a method for detecting Th17 cells.

Description

  The present invention relates to a marker for detecting IL-17-producing helper T cells (hereinafter also referred to as “Th17 cells”) and a method for detecting Th17 cells.

  Rheumatoid arthritis (hereinafter also referred to as “RA”) is a systemic inflammatory autoimmune disease whose main clinical manifestation is arthritis. Although the disease state of RA is determined by visual techniques based on the degree of pain and swelling of joints, findings by bone X-rays, and subjective symptoms, a quantitative index has not been established. Furthermore, at present, no quantitative method has been established for continuously monitoring the effect of treatment on RA.

Details of the pathogenesis of RA have not yet been clarified, but it is thought that inflammation of joint tissues occurs through a complex network of immune cells and cytokines triggered by bacterial infections.
The helper T cell group plays a central role in the immune response. Naive T cells, which are immature helper T cells, differentiate into helper T cells when antigens are presented from antigen-presenting cells. At this time, naive T cells are stimulated by specific cytokines, so that naive T cells are classified into four types. Differentiation is induced in cell types. The four cell types are helper T cells (Th1 cells) that produce interferon (IFN) -γ, helper T cells (Th2 cells) that produce interleukin (IL) -4, and helpers that produce IL-17. These are T cells (Th17 cells) and regulatory T cells (Treg cells) having an immunosuppressive effect.

  Of these helper T cells, Th17 cells have been shown to be involved in the development of RA.

  For example, in JP 2000-186046 (Patent Document 1), the amount of IL-17 produced by Th17 cells is significantly higher in the synovial fluid of RA patients than in the synovial fluid of patients with osteoarthritis. IL-17 positive cells are present in T cells in synovial tissue from RA patients, indicating that IL-17 is deeply involved in the pathogenesis of RA, particularly joint and bone destruction Yes. Furthermore, Patent Document 1 also describes that IL-17 can be used as a diagnostic marker for RA.

  JP-A-2007-506100 (Patent Document 2) analyzed cytokines in peripheral blood serum from RA patients, and found that IFN-γ, IL-1β, TNF-α, G-CSF, GM- The levels of CSF, IL-6, IL-4, IL-10, IL-13, IL-5 and IL-7 are significantly higher in RA patients, IL-2, CXCL8 / IL-8, IL- It is described that 12 and CCL2 / MCP-1 were not high values.

Regarding Th17 cells, Ivanov et al. (Cell, 2006, 126, p. 1121-1133: Non-Patent Document 1), Stumhofer et al. (Nature Immunology, 2006, vol. 7, p. 937-945: Non-Patent Document 2), Wilson (Nature Immunology, 2007, vol. 8, p.950-957: Non-Patent Document 3) and the like have revealed the following.
-A nuclear receptor called RORγt plays an important role in the differentiation of Th17 cells.
-IL-6, IL-23 and TGF-β induce differentiation of naive T cells into Th17 cells.
-Th17 cells express IL-17A, IL-17F, IL-6, IL-22, IL-26, TNF, IFN-γ and CCL20.
-IL-17 receptor and IL-12 receptor β are present on the surface of Th17 cells.

In Non-Patent Documents 1 to 3, the amount of IL-17 is measured by ELISA (enzyme-linked immunosorbent method) using an antibody specific for IL-17.
However, not only by measuring the amount of IL-17, but also by establishing a method to detect Th17 cells themselves, it is considered that the relationship between Th17 cells and immune diseases such as RA can be understood more deeply. .

JP 2000-186046 A JP 2007-506100 A

Ivanov et al., "The Orphan Nuclear Receptor RORγt Directs the Differentiation Program of Proinflammatory IL-17 + T Helper Cells", Cell, 2006, 126, p.1121-1133 Stumhofer et al., "Interleukin 27 negatively regulates the development of interleukin 17-producing T helper cells during chronic inflammation of the central nervous system" Nature Immunology, 2006, vol.7, p.937-945 Wilson et al., "Development, cytokine profile and function of human interleukin 17-producing helper T cells" Nature Immunology, 2007, vol.8, p.950-957

  The present inventors aimed to identify molecular markers that make it possible to specifically detect Th17 cells.

  The present inventors first identified a gene that is specifically expressed in Th17 cells obtained by differentiating naive T cells isolated from mouse spleen. Next, the present inventors found that the gene identified in this way is highly elevated in three types of disease model mice, such as arthritis, inflammatory bowel disease and encephalomyelitis, which are autoimmune diseases thought to be associated with Th17 cells. The gene to be expressed was identified and the present invention was completed.

Thus, the present invention
Id2 (inhibitor of DNA binding 2), Msc (musculin), Nfat5 (nuclear factor of activated T-cells 5), Nfkbiz (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, zeta), Plekho1 (pleckstrin homology domain containing, family O member 1), Runx2 (runt related transcription factor 2), Tcf12 (Transcription factor 12), Vax2 (ventral anterior homeobox containing gene 2), Zc3h12a (zinc finger CCCH type containing 12A), Tnik (TRAF2 and NCK interacting kinase), B3gnt8 UDP-GlcNAc: betaGal (beta-1,3-N-acetylglucosaminyltransferase 8), Gcnt2 (glucosaminyl (N-acetyl) transferase 2, I-branching enzyme), 1190003J15Rik (RIKEN cDNA 1190003J15 gene), Cybb (cytochrome) b-245, beta polypeptide), Rab32 (RAB32, member RAS oncogene family), Bcl2a1 (B-cell leukemia / lymphoma 2 related protein A1), Bcl7a (B-cell CLL / lymphoma 7A), Cmah (cytidine monophospho-N- acetylneuraminic acid hydroxylase), Dab2 (disabled homolog 2) Fcer1a (Fc receptor, IgE, high affinity I, alpha polypeptide), Nlrp4c (NLR family, pyrin domain containing 4C), Rbp1 (retinol binding protein 1, cellular), Stab1 (stabilin 1), Tubb2c (tubulin, beta 2c), Upp1 (uridine phosphorylase 1), Rc3h2 (ring finger and CCCH-type zinc finger domains 2), Ddx6 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 6), Actr1a (ARP1 actin-related protein 1 homolog A) or A gene encoding a protein (intracellular protein) present in a cell represented by Cyp1b1 (cytochrome P450, family 1, subfamily b, polypeptide 1);

March2 (membrane-associated ring finger (C3HC4) 2), Abca1 (ATP-binding cassette, sub-family A (ABC1), member 1), Ccr2 (chemokine (CC motif) receptor 2), Cd160 (CD160 antigen), Cd1d2 (CD1d2 antigen), Cd300a (CD300A antigen), Clec4n (C-type lectin domain family 4, member n), Cnr2 (cannabinoid receptor 2 (macrophage)), Cr1l (complement component (3b / 4b) receptor 1-like), Crtam (cytotoxic and regulatory T cell molecule), Csf2rb (colony stimulating factor 2 receptor, beta, low-affinity (granulocyte-macrophage)), Cxcr6 (chemokine (CXC motif) receptor 6), Cd163l1 (CD163 molecule-like 1), Fcgr2b (Fc receptor, IgG, low affinity IIb), H2-Ea (histocompatibility 2, class II antigen E alpha), Il27ra (interleukin 27 receptor, alpha), Itgae (integrin, alpha E, epithelial-associated), Klrb1f (killer cell lectin-like receptor subfamily B member 1F), Klrc1 (killer cell lectin-like receptor subfamily C, member 1), Klrc2 (kill er cell lectin-like receptor subfamily C, member 2), Klrd1 (killer cell lectin-like receptor, subfamily D, member 1), Pdpn (podoplanin), Slc2a6 (solute carrier family 2 (facilitated glucose transporter), member 6), Tmem176a (transmembrane protein 176A), Tnfrsf14 (tumor necrosis factor receptor superfamily, member 14 (herpesvirus entry mediator)), Tnfrsf25 (tumor necrosis factor receptor superfamily, member 25), Umodl1 (uromodulin-like 1), Vcam1 (Vascular cell adhesion molecule) 1) a gene encoding a cell membrane protein represented by Ebi2 (Epstein-Barr virus induced gene 2), Tmem176b (transmembrane protein 176B) or Il7r (interleukin 7 receptor alpha chain);

Acpp (acid phosphatase, prostate), Bmp1 (bone morphogenetic protein 1), Bpil2 (bactericidal / permeability-increasing protein-like 2), Ccl3 (chemokine (CC motif) ligand 3), Crispld2 (cysteine-rich secretory protein LCCL domain containing 2), Ctsc (cathepsin C), Gzmc (granzyme C), Il2 (interleukin 2), Lum (lumican), Ly86 (lymphocyte antigen 86), Lyz1 (lysozyme 1), Mcpt1 (mast cell protease 1), Mmp10 (matrix metallopeptidase 10), Mmp13 (matrix metallopeptidase 13), Prg4 (proteoglycan 4 (megakaryocyte stimulating factor, articular superficial zone protein)), Rln1 (Relaxin 1), S100a8 (S100 calcium binding protein A8 (calgranulin A)), Tgfbi (transforming growth) factor, beta induced), Timp1 (tissue inhibitor of metalloproteinase 1) or Apol7b or Apol7e (apolipoprotein L 7b or apolipoprotein L 7e), a gene encoding a secreted protein; or

1200015M12Rik (RIKEN cDNA 1200015M12 gene), 1200016E24Rik (RIKEN cDNA 1200016E24 gene), 1300007F04Rik (RIKEN cDNA 1300007F04 gene), 2010002N04Rik (RIKEN cDNA 2010002N04 gene), 2900073C17Rik (RIKEN cDNA 2900073C17 gene), 4930480G23Rik (24) RIKEN cDNA 4933424M12 gene), 5330403D14Rik (RIKEN cDNA 5330403D14 gene), 5430434G16Rik (RIKEN cDNA 5430434G16 gene), 5830444B04Rik (RIKEN cDNA 5830444B04 gene), 9630026M06Rik (RIKEN cDNA 9630026M06 gene), A630023P12Rik (RIKEN cDNA, RI63 cDNA) AI426953), AU015263 (expressed sequence AU015263), C030044O21Rik (RIKEN cDNA C030044O21 gene), C230085N15Rik (RIKEN cDNA C230085N15 gene), Car13 (carbonic anhydrase 13), Hmga2-ps1 (high mobility group AT-hook 2, pseudogene 1), LOC552902 (Hypothetical LOC552902), Rbm10 (RNA binding motif protein 10), Rrad (Ras-related associated with diabetes), Sbno2 (straw berry notch homolog 2), Speer1-ps1 (spermatogenesis associated glutamate (E) -rich protein 1, pseudogene 1), AK086919 (expressed sequence AK086919), BG071091 (expressed sequence BG071091), AK037075 (expressed sequence AK037075), AK048317 (expressed sequence) AK048317), AK081530 (expressed sequence AK081530), AK143436 (expressed sequence AK143436), AW538350 (expressed sequence AW538350), AK138895 (expressed sequence AK138895), AI593283 (expressed sequence AI593283), BM219171 (expressed sequence BM219171), BB204715 (expressed sequence BB204 ), AI447357 (expressed sequence AI447357), AK047185 (expressed sequence AK047185), AW491352 (expressed sequence AW491352), AK037546 (expressed sequence AK037546), BG072223 (expressed sequence BG072223), BB283635 (expressed sequence BB283635), BB800733 (expressed sequence BB800733) , AK136422 (expressed sequence AK136422), BB309694 (expressed sequence BB309694), AI661 001 (expressed sequence AI661001), AK080134 (expressed sequence AK080134), AA982044 (expressed sequence AA982044), AI447438 (expressed sequence AI447438), BB204715 (expressed sequence BB204715), AK041551 (expressed sequence AK041551), AI448729 (expressed sequence AI448729), BE951087 (Expressed sequence BE951087), AK157595 (expressed sequence AK157595), AK085158 (expressed sequence AK085158), AK028862 (expressed sequence AK028862), BG076280 (expressed sequence BG076280), BM115076 (expressed sequence BM115076), AK037590 (expressed sequence AK000000090, 77 expressed sequence GENSCAN00000037577), AK037171 (expressed sequence AK037171), AK141370 (expressed sequence AK141370), AK031033 (expressed sequence AK031033), AW120969 (expressed sequence AW120969), AI605450 (expressed sequence AI605450), AK138320 (expressed sequence AK138320), AK149443 (ed) sequence AK149443), AK090273 (expres sed sequence AK090273), BB043576 (expressed sequence BB043576) or AK036007 (expressed sequence AK036007), which is a polynucleotide derived from at least one gene selected from IL-17 producing helper T cells (Th17 cells) Polynucleotide marker for use.

The present invention also provides a protein marker for detecting Th17 cells comprising a protein encoded by at least one of the above genes.
Furthermore, the present invention relates to a method for detecting Th17 cells, comprising detecting the presence of at least one Th17 cell detection polynucleotide marker or at least one Th17 cell detection protein marker in a sample containing cells. I will provide a.
The marker of the present invention can detect Th17 cells if the presence of at least one marker is detected, but it is considered that Th17 cells can be detected more reliably by detecting a plurality of markers.

  By detecting the polynucleotide marker or protein marker of the present invention, Th17 cells can be specifically detected. Therefore, Th17 cells can be isolated by using the marker of the present invention. For example, by using the marker of the present invention, it is possible to specifically detect Th17 cells in a sample containing cells such as tissues collected from a patient, so that the patient can have RA, inflammatory bowel disease, multiple sclerosis. It is possible to find a possibility of suffering from an autoimmune disease thought to involve Th17 cells.

  In addition, the expression level of the polynucleotide marker or protein marker of the present invention is considered to be different in each stage such as the initial stage, extreme stage, and recovery stage of the autoimmune disease. Therefore, the disease state can be monitored by measuring the expression level of the marker of the present invention using ELISA, flow cytometry (FCM), microarray, or the like.

It is a figure which shows the expression level of Il7r gene in each cell of Th1, Th2, Treg, and Th17 derived from C57 / BL6 mouse. It is a figure which shows the TIMP-1 protein density | concentration in the culture supernatant of each cell of Th1, Th2, Treg, and Th17 differentiated and cultured from BALB / c mouse-derived naive T cells. It is a two-dimensional distribution map obtained by analyzing Th17 cells with a flow cytometer using anti-CD4 antibody, anti-TNFRSF14 antibody, anti-IL7R antibody, anti-PDPN antibody, and anti-CCR6 antibody.

  The polynucleotide marker for detecting Th17 cells of the present invention is a polynucleotide derived from the above gene, that is, the above gene itself (DNA), mRNA, cDNA or cRNA. The polynucleotide marker is selected from a polynucleotide derived from the gene, a mutant type and a fragment thereof.

The polynucleotide marker is a polynucleotide found to be specifically present in Th17 cells as compared to Th1, Th2 and Treg cells, or a mutant or fragment thereof. Furthermore, it was found that the above-mentioned polynucleotide marker is specifically expressed in a model mouse of a disease involving Th17 cells. Therefore, by detecting at least one of the polynucleotide markers, Th17 cells can be distinguished from Th1, Th2 and Treg cells and specifically detected.
The present invention also includes the use of a polynucleotide derived from the above gene and a protein encoded by the above gene as a marker for detecting Th17 cells.

  In the present specification, the “gene” has the same meaning as that generally used in the art, and is a part on the genome that is transcribed into mRNA and translated into protein.

  In this specification, the expression that a polynucleotide is "specifically expressed" in Th17 cells means that the expression level of the polynucleotide in Th17 cells is higher than the expression level of the polynucleotide in cells other than Th17 cells. Means significantly higher. Specifically, it means that the expression level of the polynucleotide in Th17 cells is about 1.5 times or more, more preferably 3 times or more the expression level of the polynucleotide in cells other than Th17 cells. More preferably, the expression level of the polynucleotide in Th17 cells is about 1.5 times or more, more preferably about 3 times or more the expression level of the polynucleotides in Th1 cells, Th2 cells and Treg cells.

  In the present specification, the expression that a certain polynucleotide is “specifically expressed” in a disease model mouse means that the expression of the polynucleotide in a tissue of a disease model mouse is higher than the expression level of the polynucleotide in the tissue of a healthy mouse. Meaning that the amount is significantly higher. Specifically, it means that the expression level of the polynucleotide in the tissue of a disease model mouse is about 2 times or more, more preferably 3 times or more of the expression level of the polynucleotide in the tissue of a healthy mouse.

  The base sequence of the polynucleotide marker of the present invention is already known. These can be known, for example, from Unigene or Nucleotide (both databases provided by the National Center for Biotechnology Information (NCBI) of the National Library of Medicine). Information on the base sequence of the polynucleotide marker of the present invention can be obtained from the above database using, for example, the code numbers described in “Annotation Mapped Transcripts” in Table 4 below.

  In the present specification, the “mutant form” of a polynucleotide means a polynucleotide into which a mutation that does not change the property of the protein encoded by the gene is introduced. Such mutations include deletion or substitution of one or more nucleotides from the known base sequence of the above gene, or addition of one or more nucleotides.

The above mutants usually have at least 80%, more preferably at least 85%, still more preferably at least about 90%, particularly preferably at least 95% homology with the known base sequences of the above genes.
In the present specification, “homology” of a base sequence and an amino acid sequence uses BLASTN, BLASTP, BLASTX or TBLASTN (for example, available from http://www.ncbi.nlm.nih.gov) under standard settings. Means the value calculated by

  Th17 cells can also be detected by detecting a protein encoded by the gene as a polynucleotide marker of the present invention. Therefore, a Th17 cell detection protein marker comprising a protein encoded by at least one of the above genes is also one aspect of the present invention.

  Information on the amino acid sequence of such a protein marker can be obtained based on the nucleotide sequence of the polynucleotide marker obtained from Unigene or the like. It can also be obtained from the database provided by the NCBI.

The protein marker for detecting Th17 cells may be selected from at least one protein encoded by the above gene, a functionally equivalent variant thereof, and a fragment thereof.
The “functionally equivalent mutant” of the above protein means a protein into which a mutation that does not change the function of the above protein is introduced. Such mutations include deletion or substitution of one or more amino acids from the amino acid sequence of the above known proteins, or addition of one or more amino acids.
Functionally equivalent variants of the above proteins typically have at least 80%, more preferably at least 85%, even more preferably at least about 90%, particularly preferably at least 95% of the known amino acid sequence of each of the above proteins. % Homology.

  Molecules that can specifically hybridize to the above-described polynucleotide markers can be used to detect the above-described markers, and thus are useful as probes for detecting Th17 cells. The probe may be any of nucleic acid probes such as DNA and RNA that can specifically hybridize to the polynucleotide marker, and peptide probes. As the Th17 cell detection probe, a nucleic acid probe for detecting a polynucleotide marker, particularly a DNA probe, is preferable.

In the present specification, the “molecule capable of specifically hybridizing” to a polynucleotide marker means a molecule capable of forming a duplex with the polynucleotide marker under stringent conditions.
In the present specification, the “stringent condition” means that a Th17 cell detection probe can sufficiently detect a target polynucleotide marker on a target polynucleotide marker more than a polynucleotide other than the target polynucleotide marker (for example, at least background). (Over 2 times). Stringent conditions are usually sequence-dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5 ° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. This Tm is the temperature at which 50% of the probes complementary to the target polynucleotide sequence equilibrate and hybridize under a defined ionic strength, pH and nucleic acid composition.

  Such a condition may be a condition used for hybridization between polynucleotides in a conventionally known hybridization method between polynucleotides, for example, PCR method, microarray method, Southern blotting method and the like. Specifically, the pH is 7.0 to 9.0, the salt concentration is lower than about 1.5 M Na ion, more specifically about 0.01 to 1.0 M Na ion concentration (or other salt), and the condition of at least about 30 ° C. is Can be mentioned. More specifically, for example, stringent conditions in the microarray method include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37 ° C., and 0.1 × SSC at 60-65 ° C. Includes cleaning. Stringent conditions in the PCR method include conditions of pH 7-9, 0.01-0.1 M Tris HCl, 0.05-0.15 M K ion concentration (or other salt), and at least about 55 ° C.

The sequence of the nucleic acid probe for detecting Th17 cells can be appropriately determined by those skilled in the art so that it can specifically hybridize to the polynucleotide marker based on the common general knowledge and the sequence of the polynucleotide marker.
The above nucleic acid probe for detecting Th17 cells is, for example, a generally available primer design software (for example, Primer3 (available from http://frodo.wi.mit.edu/cgi-bin/primer3/primer3.cgi), DNASIS Pro (Hitachi Software Engineering Co., Ltd.))

  The above nucleic acid probe for detecting Th17 cells can be prepared by a polynucleotide synthesis method known in the art.

The above Th17 cell detection nucleic acid probe may be labeled with a labeling substance usually used in the art. By using a labeled nucleic acid probe for detecting Th17 cells, detection of a polynucleotide marker for detecting Th17 cells, that is, detection of Th17 cells can be easily performed.
The labeling substance may be a labeling substance usually used in the art, such as a radioisotope such as ³² P, a fluorescent substance such as fluorescein, an enzyme such as alkaline phosphatase and horseradish peroxidase, and biotin.

Th17 cells can be specifically detected by using only one kind of Th17 cell detection nucleic acid probe or a combination of plural kinds.
The nucleic acid probe for detecting Th17 cells may be a set of two or more primers for amplifying the polynucleotide marker by PCR, for example.

A Th17 cell detection microarray in which the above-described nucleic acid probe is immobilized on a solid phase capable of binding to a nucleic acid is also one aspect of the present invention.
Such microarray fabrication methods are known in the art. That is, a solid phase that can bind to nucleic acid (eg, polystyrene) or a solid phase that has been surface-treated so that it can bind to nucleic acid, preferably a functional group having a cation (eg, amino group, aldehyde group, epoxy group) And the like, and a method of spotting a desired nucleic acid probe on a solid phase that has been surface-treated so as to have a surface, and a method of synthesizing a nucleic acid probe of a desired sequence on the solid phase. Another example is a method in which an activated esterified carboxyl group is introduced on the surface of a solid phase and a nucleic acid having an amino group introduced at the terminal is reacted therewith.

  The nucleic acid probe to be bound to the microarray may be labeled with a labeling substance that makes it possible to electrically or optically detect hybridization with a target gene in a sample. Such labels are known in the art.

  Molecules that can specifically bind to the above protein markers can be used to detect the above markers and are therefore useful for detecting Th17 cells. Such a molecule may be any of DNA, RNA and other nucleic acid aptamers that can specifically bind to a protein marker, and an antibody, but more preferably an antibody. In addition, when the Th17 cell-specific marker is an enzyme, it can be detected by causing a substrate to act on the enzyme to cause color development, luminescence, fluorescence, or the like.

  The above-mentioned antibody for detecting Th17 cells can be prepared, for example, by the following conventionally known procedure. Based on the nucleotide sequence of the polynucleotide marker gene or the amino acid sequence of the protein marker, a DNA molecule encoding a protein having the amino acid sequence of the protein marker is introduced into an appropriate expression vector. The obtained expression vector is introduced into an appropriate host cell, and the resulting transformed cell is cultured to obtain the target protein. The obtained protein is purified to be an immunogen, and an appropriate mammal, such as a rat or a mouse, is immunized with the immunogen and, if desired, an adjuvant. From the spleen cells of the immunized animal, antibody-producing cells that produce antibodies against the target immunogen are selected by screening. The obtained antibody-producing cells are fused with myeloma cells to obtain hybridomas, which are screened to obtain antibody-producing hybridomas that produce antibodies having specific binding properties to the proteins encoded by the above genes. be able to. The desired antibody can be obtained by culturing the obtained antibody-producing hybridoma.

  The nucleic acid aptamer that can be used to detect the above Th17 cells can be prepared by, for example, the following conventionally known procedure. A nucleic acid library having a random nucleic acid base sequence can be prepared by a known method, and aptamers that can specifically bind to a target protein (the above protein marker) can be selected by in vitro evolution (SELEX method). it can.

Molecules that can specifically bind to the above-described protein marker for detecting Th17 cells may be labeled with a labeling substance usually used in the art. By using a labeled antibody for detecting Th17 cells, detection of a protein marker for detecting Th17 cells, that is, detection of Th17 cells can be easily performed.
The labeling substance may be a labeling substance usually used in the art, such as a radioisotope such as ³² P, a fluorescent substance such as fluorescein, an enzyme such as alkaline phosphatase and horseradish peroxidase, and biotin.

A method for detecting Th17 cells by detecting the presence of at least one of the above-mentioned Th17 cell detection polynucleotide marker or Th17 cell detection protein marker in a sample containing cells is also one aspect of the present invention.
In the method of the present invention, examples of the sample containing cells include biological samples collected from mammals including humans or samples containing artificially cultured cells. Examples of biological samples include blood, tissue, joint fluid, cerebrospinal fluid, pleural effusion, ascites and the like.

An embodiment of a method for detecting the presence of the above polynucleotide marker is described.
First, nucleic acid (DNA or RNA) is extracted from a sample containing cells by a method known in the art using phenol extraction and ethanol precipitation, a commercially available DNA extraction kit, or the like.
Next, the presence of the above-described polynucleotide marker in the obtained nucleic acid sample is detected. In this detection, the above-described nucleic acid probe for detecting Th17 cells is preferably used.
The above-mentioned polynucleotide markers include PCR method, RT-PCR method, real-time PCR method, nucleic acid amplification method such as LAMP (Loop-mediated isothermal amplification) method, Southern hybridization, Northern hybridization, FISH (fluorescence in situ hybridization) ) Can be detected by a method known in the art such as a hybridization method and a microarray method. These methods are performed under the above stringent conditions, and the presence of the polynucleotide marker is detected by detecting that the above-mentioned nucleic acid probe for detecting Th17 cells has formed a hybrid by detecting the above-mentioned labeling substance. It can be detected.

  Moreover, the presence of the above-mentioned polynucleotide marker in a nucleic acid sample can also be detected by using the above-mentioned microarray.

  Next, an embodiment of a method for detecting the presence of the above-described protein marker for detecting Th17 cells will be described. For example, when the protein marker to be detected is a protein present inside the cell, the protein is extracted from the cell using a method known in the art. Extraction of proteins from cells can be performed by a known method such as disruption of cells by ultrasonic waves or solubilization using a cell lysate. And the said protein marker in the obtained protein sample is detectable using the molecule | numerator couple | bonded specifically with a protein marker. Specifically, protein markers can be detected by methods known in the art such as enzyme-linked immunosorbent assay (ELISA) and Western blotting. In detection, the above-mentioned antibody for detecting Th17 cells is preferably used as a molecule that specifically binds to a protein marker.

  For example, when the protein marker to be detected is a secreted protein, the protein marker secreted in the sample containing the cells can be detected using a molecule that specifically binds to the protein marker. In addition, cells (lymphocytes) are collected from the sample containing the above cells and collected using anti-CD3 antibody, anti-CD28 antibody, concanavalin A, PHA (phytohemagglutinin), PMA (phorbol myristate acetate), ionomycin, etc. Stimulates damaged cells. A secreted protein marker can then be detected using a molecule that specifically binds to the protein marker. Specifically, protein markers can be detected by methods known in the art such as ELISA and Western blotting. In detection, the above-mentioned antibody for detecting Th17 cells is preferably used as a molecule that specifically binds to a protein marker.

  For example, when the protein marker to be detected is a protein present on the surface of a cell, the protein marker present on the surface of the cell in the sample containing the cell is detected using a molecule that specifically binds to the protein marker. can do. In addition, a membrane fraction of a cell can be collected from a sample containing the cells, and the protein marker in the obtained membrane fraction can be detected using a molecule that specifically binds to the protein marker. Specifically, it can be detected by a method known in the art such as enzyme-linked immunosorbent assay (ELISA) or Western blotting. Further, when the protein marker to be detected is a protein present on the surface of a cell, it can also be detected by a method based on flow cytometry (FCM). In detection, the above-mentioned antibody for detecting Th17 cells is preferably used as a molecule that specifically binds to a protein marker.

For example, when a protein marker is detected by FCM, detection can be performed by the following procedure.
First, a sample containing cells is brought into contact with the above-mentioned antibody for detecting Th17 cells labeled with an appropriate labeling substance. If present, Th17 cells bind to the labeled antibody on the cell surface. Therefore, Th17 cells can be detected by passing a sample containing cells bound to the labeling substance through a flow cytometer. If desired, Th17 cells bound to a labeling substance can be discriminated and / or sorted using a cell sorter.
Such FCM methods are known per se to those skilled in the art, and the reaction conditions are appropriately determined by those skilled in the art.

Examples The present invention will be described in detail by examples, but the present invention is not limited to these examples.
Example 1
In this example, first, genes specifically expressed in cultured Th17 cells were selected by microarray expression analysis. Next, among the selected genes, genes specifically expressed in three types of disease model mice (arthritis, inflammatory bowel disease and encephalomyelitis) were identified by expression analysis by real-time PCR.

(1) Expression analysis in cultured Th17 cells
1-1. Isolation of naive T cells from mouse spleen
BALB / c mouse spleen was removed to obtain a sample containing spleen cells. After hemolysis of red blood cells in the sample using ammonium chloride, the cell fraction of CD8, B cells, monocytes, macrophages, granulocytes and erythroblasts is removed from the sample using magnetic beads (Polyscience) CD4 positive (CD4 ⁺ ) T cells were crudely purified. From the obtained CD4 ⁺ T cells, the fraction of naive T cells (CD4 ⁺ / CD25 ^neg / CD44 ^low / CD62 ^high ) was purified by sorting using a flow cytometer. Similarly, naive T cells were purified from spleen cells of C57 / BL6 mice.

1-2. Differentiation culture from naive T cells to Th1, Th2, Treg and Th17 cells The naive T cells derived from BALB / c mice obtained in the above were seeded in a 24-well plate coated with an anti-CD3 antibody at a cell density of 0.5 to 2.0 × 10 ⁶ cells / 2 ml / well. T cell medium (PRMI1640, 10% fetal bovine serum (FBS), 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamic acid, 50 μM 2-supplemented with each cytokine and antibody shown in Table 1 and anti-CD28 antibody The cells were cultured in mercaptoethanol, 100 U / ml penicillin, 100 mg / ml streptomycin) in a 37 ° C., 5% CO ₂ incubator. Three days after the start of the culture, the cytokines and antibodies shown in Table 1 were added to the medium, and further cultured for 2 to 11 days. In this way, differentiation from BALB / c mouse-derived naive T cells into Th1, Th2, Treg and Th17 cells was induced.
Similarly, the above 1. C57 / BL6 mouse-derived naive T cells obtained in the above were induced to differentiate into Th1, Th2, Treg and Th17 cells.

1-3. Confirmation of cell differentiation by flow cytometer 2. The cells were stimulated by adding phorbol myristate acetate (PMA; 50 ng / ml) and ionomycin (1 μM) to the cell suspension containing the cells differentiated and cultured as described above (each 2.5 × 10 ⁵ cells). . After 4 hours, Brefeldin A (10 μg / ml) was added, and the cells were further cultured for 2 hours. Thereafter, each cell was washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde. After fixation, each cell was treated with saponin buffer (0.5% saponin, 0.5% bovine serum albumin (BSA), 1 mM sodium azide (in PBS)) to enhance the permeability of the cell membrane. Then, the cells were reacted with anti-IFN-γ antibody, anti-IL-4 antibody and anti-IL-17 antibody. After the reaction, each cell was washed with saponin buffer and then with PBS containing 0.5% BSA, and analyzed using FACS Canto II (BD Bioscience) to differentiate into Th1, Th2, Treg and Th17 cells. confirmed.

1-4. Extraction of total RNA 2. Each cell of Th1, Th2, Treg and Th17 derived from BALB / c mice cultured for 5 days in PBS was washed with PBS, centrifuged, pelleted, and stored frozen at -80 ° C. Total RNA was extracted from the pellet using RNeasy Plus Mini Kit (QIAGEN) and stored at −80 ° C. until analysis. Similarly, the above 2. Total RNA was extracted from each cell of Th1, Th2, Treg and Th17 derived from C57 / BL6 mice cultured for 5 days.
The total RNA extraction was performed according to the description in the package insert of the kit.

1-5. Microarray expression analysis
Using One-Cycle Target Labeling and Control Reagents (Affymetrix), 4. The total RNA (1-5 μg) extracted in step 1 was reverse transcribed into cDNA and further subjected to a transcription reaction to biotinylated cRNA. 15 μg of biotinylated cRNA was added to GeneChip Mouse Genome 430 2.0 Array (Affymetrix), and hybridization was performed in GeneChip Hybridization Oven 640 (Affymetrix) for 16 hours at 45 ° C. and 60 rpm. After completion of hybridization, a microarray (DNA chip) that has been washed and fluorescently labeled using GeneChip Fluidic Station 450 (Affymetrix) is scanned using GeneChip Scanner 3000 7G (Affymetrix) to acquire fluorescence intensity data. did.
In addition, each said operation was performed according to the statement of the package insert of each reagent and apparatus.

1-6. Selection of genes specifically expressed in mouse Th17 cells Based on the fluorescence intensity data obtained in step 1, the data was standardized using expression analysis software Array Assist (Medivic Group, Inc.). Then, the relative fluorescence intensity was calculated by dividing the fluorescence intensity of each gene by the fluorescence intensity of the glyceraldehyde 3-phosphate dehydrogenase (Gapdh) gene, which is one of the housekeeping genes. The relative fluorescence intensity of each gene of Th17 cells was compared with that of Th1, Th2 and Treg cells. In at least one of BALB / c mice and C57 / BL6 mice, 128 genes whose relative fluorescence intensity in Th17 cells was 3 times or more higher than any of Th1, Th2 or Treg cells were selected (results are Not shown).

(2) Preparation of disease model mice and preparation of total RNA
2-1. Preparation of SKG arthritis model mouse An arthritis model mouse was prepared by the following method.
(I) Preparation of bacterial cell component Laminarin (laminarin from Laminaria digitata, SIGMA) was dissolved in PBS (phosphate buffer) to a concentration of 150 mg / ml. Curdlan (curdlan from Alcaligenes faecalis, SIGMA) was suspended in PBS (phosphate buffer) at 50 mg / ml.
(Ii) Administration of bacterial cell components
Laminarin (30 mg / 200 μl / mouse) or curdlan (10 mg / 200 μl / mouse) prepared in (i) was administered intraperitoneally to 7-8 week old SKG arthritic mice (female). Furthermore, 4 weeks later, curdlan prepared in (i) (10 mg / 200 μl / mouse) was intraperitoneally administered.

(Iii) Determination of severity of arthritis Joint inflammation appears after 30 days after the administration of the above bacterial cell components. The severity score was determined as follows.
Score 0: Normal Score 0.1: Swelling and erythema at the finger joint site Score 1: Mild swelling and erythema at the ankle site Score 2: Total score of severe swelling and erythema limb at the limb joint site, the value is 9 or more Individuals were used for analysis.
(Iv) Extraction of joint tissue and extraction of total RNA The skin of the joint site was removed with scissors, the footpad was removed, and the ankle joint tissue was extracted. The collected ankle tissue was stored frozen in liquid nitrogen. Total RNA was extracted from frozen ankle tissue using RNeasy Plus Mini kit (QIAGEN) and QIAshredder (QIAGEN).
This total RNA extraction was performed according to the description in the package insert of each kit.

2-2. Preparation of SCID transplanted colitis model mouse (enteritis model mouse) A colitis model mouse was prepared by the following method.
(I) Preparation of naive CD4 T cells
The spleens of 8-10 week old BALB / c mice were removed to obtain samples containing spleen cells. After hemolysis of red blood cells in the sample using ammonium chloride, the cell fraction of CD8, B cells, monocytes, macrophages, granulocytes and erythroblasts is removed from the sample using magnetic beads (Polyscience) CD4 positive (CD4 ⁺ ) T cells were crudely purified (Sample A). From the obtained CD4 ⁺ T cells, the fraction of naive T cells (CD4 ⁺ / CD25 ^neg / CD44 ^low / CD62 ^high or CD4 ⁺ / CD45RB ^high cells) was purified by sorting using a flow cytometer.
(Ii) Administration of naive CD4 T cells
Sorted naive CD4 T cells (5 × 10 ⁵ cells / 300 μl / mouse) were intraperitoneally administered to CB17 / Icr-Prkdc (scid) / CrlCrlj mice (SCID mice) 8-10 weeks old.
(Iii) Confirmation of onset After the naive CD4 T cell administration, changes in mouse body weight were observed. After about 4 weeks, weight loss associated with the onset of enteritis was observed. Individuals whose body weight decreased to 80% or less of the initial body weight were determined to be individuals with severe enterocolitis.
(Iv) Intestinal Tissue Extraction and Total RNA Extraction The large intestine was removed from a severe individual, and three fragments corresponding to the ascending colon, transverse colon, and descending colon sites were separated from the large intestine and stored frozen in liquid nitrogen. Total RNA was extracted from frozen intestinal tissue using RNeasy Plus Mini kit (QIAGEN) and QIAshredder (QIAGEN).
This total RNA extraction was performed according to the description in the package insert of each kit.

2-3. Preparation of experimental encephalomyelitis (experimental allergic encephalomyelitis: EAE) (acute type) model mouse (encephalitis model mouse) An acute type EAE model mouse was prepared by the following method.
(I) Preparation of antigen emulsion Incomplete Freund's adjuvant (Difco Laboratories) and Mycobacterium tuberculosis H37Ra (Difco Laboratories) were mixed to prepare complete Freund's adjuvant (CFA) (20 mg / ml). PLP (139-151) peptide (Bio-systhesis, Inc.) (2 mg / ml, dissolved in PBS (phosphate buffer)) and CFA are mixed in equal amounts and double hub needles (double hub needle set, Techno Chemical Co., Ltd.) Using a company), the syringe was reciprocated and mixed to prepare an antigen emulsion.
(Ii) Administration of antigen emulsion
The hair of the back of 8-10 week old SJL mice (female) was shaved with clippers, and 50 μl of the antigen emulsion was injected subcutaneously into the left and right waists of the midline at 50 μl at each site using a 1 ml syringe (total 100 μl). ). On the same day, pertussis toxin Pertussis Toxin (List Biological Laboratories) (100 ng dissolved in 200 μl PBS) was intraperitoneally administered. In addition, as healthy mice for encephalomyelitis model mice, SJL mice administered with only pertussis toxin Pertussis Toxin were used.
(Iii) Determination of severity of encephalomyelitis Cerebrospinal inflammation appears on days 10 to 21 after administration of the antigen emulsion. The severity score was determined as follows.
Score 1: Complete paralysis of the tail Score 2: Partial paralysis of the hind limb Score 3: Complete paralysis of the hind limb Score 4: Paralysis of the forelimb Score 5: Individuals with a score of 2 or more were used for the moribund state and death analysis due to general paralysis.
(Iv) Cerebrospinal tissue extraction and total RNA extraction The skull was removed from the head and the brain was removed. Also, the spinal column from which the head and tail were separated was taken out, PBS was added from the caudal vertebral foramen using a syringe, and the spinal cord was removed by pressure. The collected brain and spinal cord were frozen in liquid nitrogen. The frozen cerebrospinal tissue was pulverized with a homogenizer (As One Corporation), and total RNA was extracted using RNeasy Plus Mini kit (QIAGEN) and QIAshredder (QIAGEN).
This total RNA extraction was performed according to the description in the package insert of each kit.

(3) Expression analysis of gene in disease model mice by real-time PCR Total RNA (2.0-5.0 μg) extracted from tissue samples of each disease model mouse prepared as described in (2) above was added to poly dT primer (Hokkaido System Science Co., Ltd.), random primer (Hokkaido System Science Co., Ltd.) and Superscript III reverse transcriptase (Invitrogen Corporation) were used for reverse transcription to obtain cDNA. Moreover, the primer set corresponding to each gene selected by said (1) was produced. Using the obtained cDNA as a template, using each primer set, the expression level of the gene (threshold cycle: Ct value) by Power SYBR Green PCR Master Mix (Applied Biosystems) and 7300 Real Time PCR System (Applied Biosystems) ) Was measured. Furthermore, in order to compare each sample, data correction was performed using the Gapdh gene as an internal standard gene. And the value obtained from the following formula was used as a numerical value representing the expression level of each gene.
(Expression level of each gene) = 100000 x 2 ^-x
(However, x = (each gene Ct value)-(Gapdh gene Ct value))
In addition, each said operation was performed according to the statement of the package insert of each reagent and apparatus.
The primer set was designed using Primer3 software.

Moreover, the same experiment was conducted using the following healthy mice instead of the disease model mice.
Healthy mice for arthritis model mice: 2 BALB / c mice.
Healthy mice against colitis model mice: 2 SCID mice administered intraperitoneally with CD4 T cells (sample A) before naive CD4 T cell isolation, 2 SCID mice administered intraperitoneally with medium, and 2 BALB / c mice A total of 6 individuals.
Healthy mice against encephalomyelitis model mice: 3 SJL mice to which pertussis toxin Pertussis Toxin was administered intraperitoneally.

  Furthermore, as a reference, a primer set was also designed for genes (Il23r, Il17f, and Il19) that were conventionally known to be specifically expressed in Th17 cells, and experiments were performed in the same manner as described above.

(4) Analysis For each gene whose expression level was analyzed as described in (3) above, “value of expression level in disease model mouse (A) to (C)” and “disease with respect to expression level in healthy mouse” The ratio of expression levels in model mice (A) to (C) ”was calculated as follows.
4-1. Arthritis model analysis population: 2 healthy mice, 3 arthritis model mouse analysis samples: Hind limb joint tissue (one for each individual)
(I) Calculation of ratio of expression level in arthritis model mouse to expression level in healthy mouse (A) Calculation of average expression level of 4 samples (2 individuals x left and right hindlimb joint tissue) obtained from healthy mice did.
The ratio of the expression level in the arthritis model mouse to the average expression level in the healthy mouse, “(expression level of arthritis model mouse) / (average expression level of healthy mouse)”, 6 samples (3 individuals x left and right hindlimb joint tissue) ) Calculated for each.
Regarding the ratio of the expression level calculated above, the average value of 6 samples was calculated, and this was defined as the ratio (A) of the expression level of arthritic mice to the final expression level in healthy mice.
(Ii) Calculation of expression level (A) in arthritis model mice The average expression level (Ct value) of 6 samples (3 individuals x left and right hind limb joint tissues) obtained from arthritis model mice was calculated. The expression level (A) in arthritis model mice was used.

4-2. Number of colitis model analysis individuals: 6 healthy mice, 3 colitis model mice Analysis sample: 3 sites of the large intestine corresponding to the ascending colon, transverse colon, and descending colon (i) In colitis model mice against the expression level in healthy mice Calculation of the expression level ratio (B) The average expression level of 6 healthy mice and the average expression level of 3 colitis model mice were calculated for each of the 3 large intestine sites.
The ratio “(average expression level of colitis model mice) / (average expression level of healthy mice)” of the average expression level in colitis model mice to the average expression level in healthy mice was calculated for three sites of the large intestine. The maximum ratio of the expression levels at the three sites of the large intestine calculated above was defined as the ratio (B) of the expression level of the colitis model mouse to the final expression level in healthy mice.
(Ii) Calculation of expression level (B) in colitis model mice Expression levels of 3 colitis model mice at the site where the ratio (B) of expression levels in colitis model mice is shown in (i) above Was the final expression level (B) of the colitis model mouse.

4-3. Number of encephalomyelitis model analysis individuals: 3 healthy mice, 3 encephalomyelitis model mouse analysis samples: 3 sites in total: brain, spinal cord head half, and spinal cord tail half (i) For expression in healthy mice Calculation of ratio of expression levels (C) in encephalomyelitis model mice At each of the three sites, the average expression level of 3 healthy mice and the average expression level of 3 encephalomyelitis model mice were calculated.
The ratio of the average expression level of encephalomyelitis model mice to the average expression level of healthy mice, “(average expression level of encephalomyelitis model mice) / (average expression level of healthy mice)” was calculated for each of three sites.
The maximum ratio of the expression levels at the three sites calculated above was defined as the ratio (C) of the expression level of the encephalomyelitis model mouse to the final expression level in healthy mice.
(Ii) Calculation of expression level (C) in encephalomyelitis model mouse 3 individuals of encephalomyelitis model mice at the site where the ratio of expression level (C) of encephalomyelitis model mouse was shown in (i) above The average expression level was defined as the expression level (C) of the final encephalomyelitis model mouse.

(5) Results
Among 128 genes selected as genes that are specifically expressed in Th17 cultured cells, genes whose ratio of expression in disease model mice to expression in healthy mice is twice or more (that is, (4) above) Table 2 shows the ratios (A) to (C) of the expression levels obtained in (1).
These 27 genes were identified as genes that are specifically expressed in disease model mice involving Th17 cells. Table 2 shows the expression level values (A) to (C) calculated in the above (4) and the expression level ratios (A) to (C) for the 27 genes thus identified. Show. Furthermore, the results for genes (Il23r, Il17f, and Il19) that are conventionally known to be specifically expressed in Th17 cells are shown in Table 2 as (comparison). Table 3 shows the primer sets used in (3) above for the 27 identified genes.
Of the 27 genes in Table 2, Ccl20, Il17a, Il22, and RORγt (shown in italics in Table 2) are genes that are known to be specifically expressed in Th17 cells. Therefore, 23 genes other than these 4 genes were newly identified as genes that are specifically expressed in Th17 cells and also specifically expressed in disease model mice involving Th17 cells.
The 23 genes identified this time are considered to be novel markers useful for the detection of Th17 cells. Of these genes, the higher the expression level (preferably the expression level is 1000 or more, more preferably 10,000 or more) is considered to be more promising as a marker.

Example 2
In this example, first, genes specifically expressed in Th17 cultured cells were selected by microarray expression analysis. Next, among the selected genes, genes specifically expressed in three types of disease model mice (arthritis, inflammatory bowel disease and encephalomyelitis) were identified by microarray expression analysis.

(1) Expression analysis in cultured Th17 cells Expression analysis in cultured Th17 cells was performed in the same manner as in Example 1 (1), and the expression in Th17 cells was 1.5 times that of any of Th1, Th2 and Treg cells. 586 higher genes were selected (results not shown).

(2) Expression analysis of gene in disease model mouse by microarray Next, expression analysis in a disease model mouse of the 586 gene selected in the above (1) was performed using the microarray.
Specifically, in the same manner as in Example 1 (2), tissue samples were first collected from the above three disease model mice (arthritis, enteritis, and encephalitis), and total RNA was extracted from the collected tissue samples.
Next, total RNA extracted using One-Cycle Target Labeling and Control Reagents (Affymetrix) or Two-Cycle Target Labeling and Control Reagents (Affymetrix) (1-5μg for One-cycle, for Two-Cycle) 10-100 μg) was reverse transcribed into cDNA according to the instruction manual, and further subjected to transcription reaction to biotinylated cRNA. 15 μg of biotinylated cRNA was placed in GeneChip Mouse Genome 430 2.0 Array (Affymetrix), and hybridization was performed in GeneChip Hybridization Oven 640 (Affymetrix) for 16 hours at 45 ° C. and 60 rpm. After completion of hybridization, the microarray washed and fluorescently labeled using GeneChip Fluidic Station 450 (Affymetrix) was scanned using GeneChip Scanner 3000 7G (Affymetrix) to acquire fluorescence intensity data.
Moreover, the same experiment was conducted using healthy mice as described in Example 1 instead of disease model mice.

(3) Analysis Based on the obtained fluorescence intensity data, standardize the data using expression analysis software Array Assist (Medivic Group, Inc.), and divide the fluorescence intensity of each gene by the fluorescence intensity of the Gapdh gene. Relative fluorescence units (RFU) were calculated. Thus, the relative fluorescence intensity calculated for the arthritis model mouse is the expression level (D) in the arthritis model mouse, and the relative fluorescence intensity calculated for the colitis model mouse is the expression level (E) in the colitis model mouse. The relative fluorescence intensity calculated for the encephalomyelitis model mouse was defined as the expression level (F) in the encephalomyelitis model mouse.
Next, the relative fluorescence intensity due to the expression of each gene in a healthy mouse is compared with that of a disease model mouse, and the ratio of the relative fluorescence intensity of the disease model mouse to the relative fluorescence intensity of the healthy mouse “(the relative fluorescence intensity of the disease model mouse ) / (Relative fluorescence intensity of healthy mice)]. The ratio of the relative fluorescence intensity calculated for the arthritis model mouse in this way was defined as the ratio (D) of the expression level of the arthritis mouse to the expression level of the healthy mouse. The ratio of the relative fluorescence intensity calculated for the colitis model mouse was defined as the ratio (E) of the expression level of the colitis mouse to the expression level of the healthy mouse. The ratio of the relative fluorescence intensity calculated for the encephalomyelitis model mouse was defined as the ratio (F) of the expression level of the encephalomyelitis mouse to the expression level of the healthy mouse.

(4) Results
Among the 586 genes selected as genes that are specifically expressed in Th17 cultured cells, the ratio of the expression level in the disease model mouse to the expression level in the healthy mouse is 2 times or more (that is, the above (3) Table 4 shows the ratios (D) to (F) of the expression levels obtained in (1).
These 150 genes were identified as genes that are specifically expressed in disease model mice involving Th17 cells. For the 150 genes thus identified, the measured expression level values (D) to (F) and the calculated expression level ratios (D) to (F) are shown in Table 4. Indicated. Furthermore, Table 4 also shows the Unigene code of each gene, the NCBI code indicating the amino acid sequence of the protein encoded by each gene, the gene symbol, and Annotation Mapped Transcripts.

  Of the 150 genes shown in Table 4, genes that have been known to be specifically expressed in Th17 cells are Gpr15, Ccl20, Il17a, Il21 and Il22 (shown in italics in Table 4). is there. Therefore, 145 genes other than these 5 genes were newly identified as genes that are specifically expressed in Th17 cultured cells and specifically expressed in disease model mice involving Th17 cells. The 145 genes identified this time are considered to be useful novel markers for detecting Th17 cells. Of these genes, the higher the expression level (preferably the expression level is 1000 or more, more preferably 10,000 or more) is considered to be more promising as a marker.

  From these results, similarly to the five genes shown in italics in Table 4 that are conventionally known to express each gene shown in Table 4 specifically in Th17 cells, Th17 cells and Th17 cells It was shown that it is specifically expressed in a model mouse of a disease that can be involved.

  From the above, by detecting each gene shown in Table 2 and Table 4 using a method known in the art such as PCR method, the expression of the protein encoded by these genes can be determined by ELISA method. It is considered that Th17 cells can be specifically detected by detection using a method known in the art such as a method using a microarray or a method using a flow cytometer.

Example 3
In this example, the expression level of the Il7r gene in cultured Th cells was measured using real-time PCR analysis.

1-4 of Example 1. The total RNA (2.5 μg) obtained in step 1 was reverse transcribed using poly dT primer (Hokkaido System Science Co., Ltd.), random primer (Hokkaido System Science Co., Ltd.) and Superscript III reverse transcriptase (Invitrogen Corporation). cDNA was obtained. In addition, a primer set corresponding to the Il7r gene was prepared. Then, using the obtained cDNA as a template, this primer set was used to measure the Ct value of the Il7r gene by Power SYBR Green PCR Master Mix (Applied Biosystems) and 7300 Real Time PCR System (Applied Biosystems).
In addition, each said operation was performed according to the statement of the package insert of each reagent and apparatus. The primer set was designed using Primer3 software. Table 5 shows the sequences of primer sets for the Il7r gene and the Gapdh gene.

In order to compare between samples, Ct values were similarly determined using the Gapdh gene as an internal standard gene. The value obtained from the following formula was used as a numerical value representing the expression level of the Il7r gene.
(Il7r gene expression level) = 100000 x 2 ^-x
(However, x = (Il7r gene Ct value)-(Gapdh gene Ct value))
FIG. 1 shows the expression level of the Il7r gene in Th1, Th2, Treg and Th17 cells derived from C57 / BL6 mice.

  As can be seen from FIG. 1, the Il7r gene is strongly expressed in Th17 cells. From this, it was shown that Th17 cells can be detected using the Il7r gene as a marker.

Example 4
In this example, the TIMP-1 protein concentration in the culture supernatant of Th1, Th2, Treg and Th17 cells differentiated and cultured from BALB / c mouse-derived naive T cells was measured by ELISA.

  The mouse TIMP-1 DuoSet ELISA Development kit (R & D Systems) was used for the measurement. Casein (SIGMA-Aldrich) was added to the blocking solution so that the final concentration was 0.5%, and detection was performed with a peroxidase chemiluminescence system using FEMTOGLOW Plus (Michigan Diagnostics LLC) as the luminescent substrate. Was performed as described in the package insert of the kit.

  32 samples each of Th1 and Th2 cells and 28 samples of Treg and Th17 cells were measured, and a significant difference (p value) between the measured values of each cell was determined by Mann-Whitney test. The result is shown in FIG.

  As is clear from FIG. 2, it can be seen that TIMP-1 protein is secreted in a large amount in Th17 cells. From this, it was shown that Th17 cells can be detected using TIMP-1 protein as a marker.

Example 5
In this example, analysis using a flow cytometer of a Th17 cell using an anti-TNFRSF14 antibody, an anti-IL7R antibody and an anti-PDPN antibody, and an analysis using a flow cytometer of a Th17 cell using a conventional anti-CCR6 antibody A comparison was made.

Example 1-2 1-2. In the Th1 differentiated culture, Th2, Treg and Th17 cells (10 ⁷ cells / ml, respectively), PE-Cy7-labeled anti-CD4 antibody (1.0μg / ml, BD Biosciences), PE-labeled anti-TNFRSF14 antibody (2.0 [mu] g / ml, eBioscience), FITC-labeled anti-IL7R antibody (1.0 μg / ml, Biolegend), anti-PDPN antibody (2.5 μg / ml, R & D Systems) and Alexa647-labeled anti-CCR6 antibody (5.0 μg / ml, Biolegend) Added and allowed to react for 20 minutes.
Since the anti-PDPN antibody is not fluorescently labeled, anti-goat IgG-Alexa488 antibody (1.0 μg / ml, Molecular Probes) was further reacted for 20 minutes as a fluorescently labeled secondary antibody against the anti-PDPN antibody.

  After the above reaction, Th1, Th2, Treg and Th17 cells were washed with PBS containing 0.5% BSA and then suspended in PBS containing 0.5 μg / ml 7-amino-actinomycin D (7-AAD) and 0.5% BSA. did. Then, the expression of each surface antigen in Th1, Th2, Treg and Th17 cells was confirmed by analysis using FACS CantoII (BD Bioscience) and FACS DIVA software, and the ratio (%) of the number of positive cells to the total number of cells Asked. FIG. 3 is a two-dimensional distribution diagram showing the results of analyzing Th17 cells with a flow cytometer using anti-CD4 antibody, anti-TNFRSF14 antibody, anti-IL7R antibody, anti-PDPN antibody and anti-CCR6 antibody. Table 6 shows the ratio (%) of the number of positive cells to the total number of cells for Th17 cells.

  As is clear from FIG. 3 and Table 6, the flow cytometer of Th17 cells using anti-TNFRSF14 antibody, anti-IL7R antibody and anti-PDPN antibody was compared with the analysis by conventional flow cytometer of Th17 cells using anti-CCR6 antibody. This analysis showed that Th17 cells can be detected with higher sensitivity.

  This application is related to Japanese Patent Application No. 2008-218196 filed on Aug. 27, 2008, and all of the claims, specification and abstract are incorporated herein by reference.

Claims (3)

Id2, Msc, Nfat5, Nfkbiz, Plekho1, Runx2, Tcf12, Vax2, Zc3h12a, Tnik, B3gnt8, Gcnt2, 1190003J15Rik, Cybb, Rab32, Bcl2a1, Bcl7a, Cmah, Dab2, Fcer1a, Nl4, Fcer1a, Nl1 A gene encoding an intracellular protein represented by Rc3h2, Ddx6, Actr1a or Cyp1b1;
March2, Abca1, Ccr2, Cd160, Cd1d2, Cd300a, Clec4n, Cnr2, Cr1l, Crtam, Csf2rb, Cxcr6, Cd163l1, Fcgr2b, H2-Ea, Il27ra, Itgae, Klrb1f, Klrc1d, Klrc1d, Klrc1d A gene encoding a cell membrane protein represented by Tnfrsf14, Tnfrsf25, Umodl1, Vcam1, Ebi2, Tmem176b or Il7r;
Acpp, Bmp1, Bpil2, Ccl3, Crispld2, Ctsc, Gzmc, Il2, Lum, Ly86, Lyz1, Mcpt1, Mmp10, Mmp13, Prg4, Rln1, S100a8, Tgfbi, Timp1, or Apol7b or Apol7e Or
1200015M12Rik, 1200016E24Rik, 1300007F04Rik, 2010002N04Rik, 2900073C17Rik, 4930480G23Rik, 4933424M12Rik, 5330403D14Rik, 5430434G16Rik, 5830444B04Rik, 9630026M06Rik, A630023P12Rik, AI426953, AU015263, C030044O21Rik, C230085N15Rik, Car13, Hmga2-ps1, LOC552902, Rbm10, Rrad, Sbno2, Speer1-ps1, AK086919, BG071091, AK037075, AK048317, AK081530, AK143436, AW538350, AK138895, AI593283, BM219171, BB204715, AI447357, AK047185, AW491352, AK037546, BG072223, BB283635, BB800733, AK136422, BB309694, AI661AA, 715 AK041551, AI448729, BE951087, AK157595, AK095158, AK085158, AK028862, BG076280, BM115076, AK037590, GENSCAN00000037577, AK037171, AK141370, AK031033, AW120969, AI605450, AK138320, AK149443, AK090273, BB043576 or AK036007 Polynucleotide for detecting IL-17-producing helper T cells, which are polynucleotides derived from two genes Octreotide marker.   A protein marker for detecting IL-17-producing helper T cells, comprising a protein encoded by at least one gene according to claim 1.   Detecting the presence of at least one polynucleotide marker for detecting Th17 cells according to claim 1 or at least one protein marker for detecting Th17 cells according to claim 2 in a sample containing cells. A method for detecting IL-17-producing helper T cells.

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